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Endocrinology Vol. 148, No. 8 3655-3665
Copyright © 2007 by The Endocrine Society

Androgen-Dependent Regulation of Brain-Derived Neurotrophic Factor and Tyrosine Kinase B in the Sexually Dimorphic Spinal Nucleus of the Bulbocavernosus

Erich N. Ottem, Laurel A. Beck, Cynthia L. Jordan and S. Marc Breedlove

Neuroscience Program, Michigan State University, East Lansing, Michigan 48824

Address all correspondence and requests for reprints to: Erich N. Ottem, Neuroscience Program, 108 Giltner Hall, Michigan State University, East Lansing, Michigan 48824. E-mail: ottem{at}msu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Castration of adult male rats causes the dendrites of androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) to retract. Brain-derived neurotrophic factor (BDNF), via activation of tyrosine receptor kinase B (trkB), has been implicated in mediating androgen effects on SNB dendrites. We used in situ hybridization to demonstrate that SNB motoneurons in gonadally intact adult male rats contain mRNA for both BDNF and trkB. Two weeks after gonadectomy, both transcripts were significantly decreased in SNB motoneurons but not in the non-androgen-responsive motoneurons of the adjacent retrodorsolateral nucleus (RDLN). In a second experiment, target perineal and foot muscles of SNB and RDLN motoneurons, respectively, were injected with the retrograde tracer Fluorogold, and then immunocytochemistry was performed to examine the distribution of BDNF and trkB proteins in SNB and RDLN motoneurons and their glutamatergic afferents. Confocal analysis revealed that gonadectomy induces a loss of BDNF protein in SNB dendrites but not in RDLN dendrites. Testosterone treatment of castrates prevented the loss of BDNF from SNB dendrites. Confocal analysis also revealed trkB protein in SNB and RDLN dendrites and in their glutamatergic afferents. Gonadectomy had no discernable effect on trkB protein in SNB or RDLN motoneurons or in their glutamatergic afferents. These results suggest that androgen maintains a BDNF-signaling pathway in SNB motoneurons that may underlie the maintenance of dendritic structure and synaptic signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SPINAL NUCLEUS of the bulbocavernosus (SNB) is a sexually dimorphic group of motoneurons found near the midline of the ventral horn in the lower lumbar spinal cord of rats. In males, SNB motoneurons innervate the bulbocavernosus (BC) and levator ani (LA) muscles that attach to the base of the penis and control penile reflexes and copulation (1, 2, 3). Male SNB motoneurons have highly branched dendritic arbors, the development and maintenance of which are dependent on androgens (4, 5). For example, adult SNB dendrites retract after castration, whereas androgen replacement maintains dendritic length (4, 5).

Brain-derived neurotrophic factor (BDNF) is implicated in the androgen-dependent maintenance of adult SNB morphology. Axotomy of adult SNB motoneurons decreases BDNF levels in these motoneurons, suggesting that BC/LA muscles are a source of BDNF for SNB cells (6). Axotomy also reduces significantly the size of SNB somata, independent of androgens, and BDNF treatment at the site of the cut axons reverses this effect (6). In contrast, only the combination of androgen replacement and BDNF treatment of severed axons restores SNB motoneuron dendrites (7). Thus, a BC/LA source of BDNF may be important for androgen’s ability to maintain SNB dendritic morphology, but BDNF from other sources may also play a role.

BDNF protein is found in both SNB motoneurons and the BC/LA complex (8, 9). However, it is unknown whether BDNF protein in SNB motoneurons is transported retrogradely from BC/LA muscles, or is synthesized in the motoneurons themselves, or is derived from both sources. In Yang et al. (7), axotomy presumably cut off a muscle source of BDNF, but it is possible that axotomy also perturbed BDNF expression in the motoneurons. Because restoration of SNB dendrites by BDNF also requires androgens, androgens may increase the supply of BDNF at sites other than the BC/LA muscles. SNB motoneurons and their afferents are potential sources of BDNF that may also contribute to maintaining SNB dendritic morphology. Many studies have examined BDNF protein in the spinal cord; however, only a few studies have investigated the expression of BDNF mRNA in motoneurons (10, 11, 12, 13). Although these studies suggest that many, but not all, spinal motoneurons throughout the spinal cord contain mRNA for BDNF and its receptor, tyrosine kinase B (trkB), none specifically show that SNB motoneurons contain these messages. Thus, one goal of this study is to establish whether SNB motoneurons contain BDNF mRNA and are thus likely to produce BDNF protein.

In other regions of the central nervous system, BDNF interacts with the neurotransmitter glutamate to enhance synaptic strength. For example, BDNF and glutamate can be released from the same presynaptic terminals to act at postsynaptic trkB receptors, which enhances excitatory signaling (14, 15, 16, 17). BDNF acting as a retrograde signal can also enhance glutamate release from presynaptic neurons (18, 19, 20). Glutamatergic terminals are found throughout the spinal cord (21, 22, 23), but little is known about the role that BDNF and glutamate may play together at SNB dendritic synapses. Moreover, although it is known that castration causes a significant reduction in synaptic input to SNB dendrites, we do not know specifically about glutamatergic synapses and whether BDNF and/or trkB changes there. Thus, a second goal of this study was to determine the extent of colocalization of BDNF and trkB protein with glutamatergic terminals and to determine whether castration affects the distribution of these proteins in SNB motoneurons and/or glutamatergic afferents.

We address several questions regarding the origins of BDNF protein in SNB motoneurons and possible regulation of this neurotrophin and its receptor by androgens. First, using in situ hybridization to detect BDNF mRNA, we asked whether SNB motoneurons transcribe the BDNF gene and whether levels of BDNF mRNA in SNB motoneurons are dependent on androgens. Second, we asked whether SNB motoneurons contain mRNA for the BDNF receptor, trkB, and whether androgens regulate expression of mRNA for this receptor. Third, using laser confocal microscopy, we characterized the distribution of BDNF and trkB proteins in SNB motoneurons, their proximal dendrites, and their glutamatergic afferents. Finally, we asked whether the distribution of BDNF and/or trkB proteins in adult SNB motoneurons and their glutamatergic afferents depends on the presence of androgens.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult (90-d-old) male Sprague Dawley rats (Charles River, Boston, MA) were maintained according to NIH Guidelines for the Care and Use of Laboratory Animals, and the Institutional Animal Care and Use Committee of the Michigan State University approved all treatment protocols. Animals were housed in a temperature- and light-controlled room (14-h light, 10-h dark cycle; lights on at 0500 h) with food and water provided ad libitum.

In situ hybridization (ISH) studies
To determine whether SNB motoneurons contain BDNF and/or trkB mRNAs and whether the levels of either transcript are regulated by androgens, adult (90-d-old) male rats were either sham gonadectomized (SHX; n = 7) or gonadectomized (GDX; n = 7) under isoflurane anesthesia and killed 2 wk later with CO2. Immediately after killing, the lower lumbar spinal cords were removed and frozen in powdered dry ice, wrapped in Parafilm, and stored at –80 C in sealed tubes. We obtained 12-µm transverse cryosections through the rostrocaudal extent of the lower lumbar spinal cord. Alternate sections were placed on two series of gelatin-coated Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA) for the two ISH studies; one series was used to examine BDNF mRNA expression and the other to examine trkB expression.

The cDNA template used for transcription of cRNA probes complementary to BDNF mRNA was a 563-bp fragment corresponding to bases 2203–2770 of the rat BDNF cDNA (GenBank accession no. D10938). The template was prepared using RT-PCR with a forward primer sequence of 5'-ACGGACAAGGCAACTTGGCC-3' and a reverse primer sequence of 5' TACGATTGGGTAGTTCGGCA-3'. The BDNF template corresponds to exon V of the full BDNF gene (24). Because BDNF mRNA occurs in multiple spliced variants containing one of exons I-IV coupled to exon V (25), we designed a probe complementary to the common exon to maximize BDNF mRNA detection. The cDNA template for trkB was a 731-bp fragment corresponding to bases 964-1694 of the rat trkB cDNA (GenBank accession no. NM012731). This template was prepared using a forward primer sequence of 5'-TTCCGGCTTAAAGTTTGTGG-3' and reverse primer sequence of 5'-CGTGGTACTCCGTGTGATTG-3'. Both BDNF and trkB cDNA fragments were cloned into a TOPO-TA vector (Invitrogen, Carlsbad, CA) and sequenced to verify identity. Antisense cRNA probes complementary to BDNF and trkB mRNA were transcribed using SP6 RNA polymerase (Promega, Madison, WI). We transcribed sense cRNA for BDNF and trkB with T7 RNA polymerase (Promega) to use as controls.

Sections were thawed, fixed, and prehybridized before applying 35S-labeled cRNA antisense probes for BDNF or trkB mRNAs. A number of tissue sections, sampled throughout the rostrocaudal extent of the SNB region, were reserved for hybridization with 35S-labeled sense probes for controls in each experiment. Briefly, sections were thawed for 10 min and then fixed with 4% formalin in PBS for 15 min and treated with 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCl (pH 8.0). Next, the sections were dehydrated in a series of ethanol washes, delipidated in chloroform, and then rehydrated in 95% ethanol and allowed to dry. 35S-labeled BDNF (study 1) or trkB (study 2) cRNA probes (1 x 106 cpm) were then applied to each slide in three 25-µl drops of hybridization buffer to ensure all spinal sections were covered. The hybridization buffer contained 2x standard saline citrate solution (SSC; 1x SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.2), 50% formamide (vol/vol), 15% (wt/vol) dextran sulfate, 250 µg/µl tRNA, 1x Denhardt’s solution (0.02% Ficoll, 0.02% polyvinylpyrolidine, 0.02% BSA) and 400 mM dithiothreitol (26). Tissue sections were covered with Hybri-slips (Sigma-Aldrich, St. Louis, MO) and incubated at 55 C in humid conditions overnight. After incubation, sections were washed twice for 15 min in 1x SSC on an orbital shaker. The sections were then washed twice for 20 min in 50% (vol/vol) formamide/2x SSC at 52 C, followed by two more washes in 2x SSC for 10 min each. Sections were then placed in RNase buffer (0.5 M NaCl; 10 mM Tris, pH 8.0; and 1 mM EDTA, pH 8.0) containing 25 µg/ml RNase A (Roche, Indianapolis, IN) and incubated at 37 C for 30 min with shaking. The RNase wash was followed by two rinses in 2x SSC for 10 min and a final rinse in 50% formamide/2x SSC for 20 min at 52 C.

To visualize radiolabeled antisense probes and sense controls for BDNF and trkB mRNA, sections were dipped in NTB emulsion (Kodak, Rochester, NY; diluted 1:1 with deionized distilled water). After the exposure period (10 d exposure for BDNF; 14 d exposure for trkB), the slides were developed in Dektol (Kodak) and fixed in Kodak Fixer. To visualize motoneurons, sections were stained in a 4% cresyl violet solution and destained in 70% ethanol. The slides were then coverslipped and prepared for analysis of autoradiographic signal.

Image analysis of ISH
We used Scion Image Analysis System interfaced with a Nikon Optiphot-2 light microscope (Nikon USA, Melville, NY) through a 3CCD color video camera (Optronics DEI, Goleta, CA). We analyzed both SNB motoneurons and motoneurons of the retrodorsolateral nucleus (RDLN) using a x4 oil immersion objective. The RDLN motoneurons that innervate foot muscles serve as an excellent internal control when studying the SNB because they are present in the same sections as SNB motoneurons and are relatively unresponsive to androgens (27, 28). Briefly, we first set a threshold that digitally highlighted pixels corresponding to silver grains (autoradiographic signal for BDNF and/or trkB mRNA). Using our image analysis software, we then determined the average area of an SNB or RDLN motoneuron. Using these measures and Scion Image Analysis software, we next determined average nonspecific autoradiographic background signal contained in motoneuron-size area by determining the average area of pixels (occupied by silver grains) contained in the white matter of the funiculus in each section. Because the funiculus consists of white matter, little mRNA should be present, and any autoradiographic signal should be considered nonspecific and therefore background. Using these methods, we determined the average autoradiographic background for each section. We then determined the number of highlighted pixels overlying cresyl violet-stained SNB and RDLN motoneurons. Motoneurons were considered positive for BDNF and/or trkB gene expression if the density of silver grains over the neuron was at least five times background density. All analyses were carried out blind to treatment. For each animal, 20–30 SNB and 20–30 RDLN motoneurons were sampled randomly. SNB and RDLN motoneuronal cell bodies were circumscribed using the Scion Image Analysis software, and pixels occupied by silver grains denoting BDNF and/or trkB mRNA autoradiographic signal were highlighted to threshold, as described above. The average nonspecific autoradiographic background signal determined from analysis of white matter (described above) was subtracted from the total area of pixels (occupied by silver grains), and the resulting corrected total area of pixels was calculated per circumscribed motoneuron and averaged across sampled SNB or RDLN motoneurons for each animal. These means were used to determine grand means of area covered by grains per motoneuron for each treatment group. Finally, two-tailed t tests were used to determine the effect of GDX on BDNF and trkB mRNA expression in SNB and RDLN motoneurons.

Immunofluorescence
Immunofluorescence and laser confocal microscopy was used to examine the expression pattern of BDNF and trkB proteins in SNB and RDLN motoneurons and in glutamatergic appositions to these motoneurons. Separate cohorts of animals were used to examine the expression patterns of each protein. For each study, three treatment groups of adult 90-d-old male Sprague Dawley rats were anesthetized under isoflurane. One group was SHX (n = 6 for each study) and received sc implants of two empty (blank) 2-cm-long (effective release length) Silastic capsules (1.6 mm inner diameter; 3.2 mm outer diameter). The second and third experimental groups were both GDX and implanted with either blank capsules (n = 6 for each study) or implanted with capsules containing crystalline testosterone (T; n = 6 for each study). Because BDNF has been shown to be contained in the proximal extent of neural dendrites (29, 30, 31), we chose the retrograde tracer Fluorogold to use in this experiment because of its ability to strongly label proximal motoneuron arbor (32). Although still anesthetized, all animals were injected with 5 µl Fluorogold (3% Fluorogold, 1% dimethylsulfoxide; Fluorochrome, Inc., Denver CO) into the left BC muscle to retrogradely label SNB motoneurons and an intrinsic muscle of the right rear foot to retrogradely label RDLN motoneurons. Because castration-induced reduction in SNB dendritic arbors has been shown to occur as early as 25 d after GDX (4, 7, 33, 34, 35), we assumed that the mechanisms initiating SNB dendritic retraction would occur earlier. Thus, 14 d after surgery, animals were killed with an overdose of pentobarbital and perfused transcardially with 0.9% saline, followed by 200 ml 4% (wt/vol) paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Spinal cords were removed, postfixed in 4% paraformaldehyde at 4 C for 2 h, and then placed in a solution of 30% sucrose in 0.1 M phosphate buffer at 4 C. Finally, 40-µm cross-sections of the lumbar spinal cord were obtained using a freezing sliding microtome.

Immediately after sectioning, free-floating sections were washed in PBS (140 mM NaCl, 10.7 mM KCl, 1 mM KH2PO4, 10 mM Na2HPO4, pH 7.4) and then incubated in blocking buffer (10% normal donkey serum, 0.2% Triton X-100, and 0.02% sodium azide in PBS) at room temperature for 1 h. Rabbit polyclonal anti-Fluorogold (1:1000; Chemicon, Temecula, CA) was used to visualize retrogradely Fluorogold-labeled SNB and RDLN motoneurons. To visualize excitatory terminals in these studies, we used antibodies against vesicular glutamate transporter 1 (VGLUT1), which specifically labels glutamatergic terminals (36, 37). In studies examining BDNF expression in retrogradely identified SNB and RDLN motoneurons, sections were incubated for 24 h at room temperature followed by 48 h at 4 C in blocking buffer containing a cocktail of sheep anti-BDNF (1:500; Chemicon), guinea pig polyclonal anti-VGLUT1 (1:5000; Chemicon), and the anti-Fluorogold antibody (1:1000; Chemicon). In studies examining trkB expression patterns in lower lumbar spinal cord, we used chicken polyclonal anti-trkB (1:500; Promega) combined with the anti-Fluorogold and anti-VGLUT1 in blocking buffer as described above. After incubation in primary antisera, sections were washed in PBS and incubated at room temperature for 1 h in a mixture of fluorescently labeled secondary antibodies (each of the secondary antisera was diluted 1:150 in PBS containing 0.2% Triton X-100 and 0.02% sodium azide). We detected Fluorogold with AlexaFluor 488-conjugated donkey antirabbit IgG (Molecular Probes, Portland, OR), VGLUT1 with Cy5-conjugated donkey anti-guinea pig IgG (Jackson ImmunoResearch, West Grove, PA), BDNF with Texas Red-conjugated donkey antisheep IgG (Jackson ImmunoResearch), and trkB with Texas Red-conjugated donkey anti-chicken IgG (Jackson ImmunoResearch). After incubation with secondary antibodies, sections were washed in PBS, mounted on glass slides, and allowed to air dry before applying coverslips with DPX mounting media (Fluka, Steinheim, Germany). For all experiments, signal was not detected in absence of the primary antibody or in procedures to test for cross-reactivity between multiple primary and secondary antibodies. Our findings agree with the results of previous studies that have extensively characterized the specificity of anti-Fluorogold (38, 39, 40), anti-BDNF (41, 42, 43), anti-trkB (44, 45, 46), and anti-VGLUT-1 (47, 48, 49).

Confocal analysis of immunostained tissue
Analysis was performed using a Zeiss LSM Pascal confocal microscope (Carl Zeiss International, Oberkochen, Germany). For image scans, the pinhole diameter was optimized to 1.11 Airy disk, and image size was set at 512 x 512 pixels. Confocal images were obtained using a x40 oil immersion objective (1.4 numerical aperture) and x1.5 digital zoom. After SNB or RDLN soma and dendrites were localized using epifluorescent (mercury vapor) illumination, we switched to confocal mode (laser) and performed optical sectioning of a selected SNB or RDLN motoneuron. For each motoneuron (n = 25 per animal), we obtained stacks of 35–40 optical sections (Z-thickness, 0.5 µm), depending on the orientation of the individual neuron. Each Z-series was composed of scans through three separate channels (488, 543, and 643 nm excitation) for each study. We performed scans at each wavelength to visualize each tag independently to eliminate bleed-through between individual channels. Images from single scans and compiled Z-series were captured and saved.

For localizing VGLUT1 and BDNF in or near dendritic arbors of SNB and RDLN motoneurons, we used Zeiss LSM 5 Image Browser software that allows virtual rotation of cells to verify that VGLUT1-containing appositions actually contact SNB and RDLN motoneurons. We also used the LSM Browser software to measure the length of Fluorogold-labeled dendrites containing BDNF. The length of dendrite containing Fluorogold and BDNF labels was measured from the beginning of the dendritic extension marked by the perimeter of the soma. Measurements were expressed as percentage of Fluorogold-labeled dendrite containing BDNF immunoreactivity. Mean percentages within each animal were used to calculate grand means for each treatment group. Separate one-way ANOVAs were used to determine whether androgens affect the distribution of BDNF in the dendrites of SNB and RDLN motoneurons. Post hoc comparisons were performed using Bonferroni-adjusted t tests.

We used similar analysis parameters to assess trkB expression in dendrites and VGLUT1-positive glutamatergic dendritic afferents. We determined the mean percentage of glutamatergic appositions on Fluorogold-labeled dendrites that contained trkB immunoreactivity for each animal. These means were used to calculate grand means for each treatment group. Separate one-way ANOVAs were used to determine whether androgens affected the percentage of VGLUT1-positive appositions contacting SNB and RDLN motoneuronal dendrites that were also immunopositive for trkB.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SNB and RDLN motoneurons contain BDNF and trkB mRNA
ISH indicated that all adult SNB and RDLN motoneurons contain BDNF and trkB mRNA. For both the BDNF and trkB probes, the density of silver grains was selectively and consistently increased over the somas of SNB and RDLN motoneurons relative to background levels in both GDX and gonadally intact male rats [Fig. 1Go, A and B (BDNF mRNA) and D and E (trkB mRNA)]. All SNB and RDLN motoneurons observed met the criteria of containing BDNF and trkB mRNA autoradiographic signal that was five times above background levels. We found no autoradiographic labeling in sense-strand control sections, indicating that the BDNF cRNA probe is specific for BDNF mRNA (Fig. 1CGo) and the trkB probe is specific for trkB mRNA (Fig. 1FGo). Because all SNB or RDLN motoneurons sampled in each experiment were observed to be positive for either BDNF or trkB mRNA autoradiographic signal, these results suggest that the number of BDNF and trkB mRNA-expressing motoneurons is not influenced by adult gonadal androgens.


Figure 1
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  		FIG. 1. SNB and RDLN motoneurons contain BDNF and trkB mRNA. BDNF mRNA were detected with 35S-labeled cRNA probes and are shown as an accumulation of black silver grains over a cresyl violet-stained cell body. All SNB (A) and RDLN (B) motoneurons observed show such accumulation of silver grains and thus express BDNF mRNA. A comparable accumulation of silver grains over SNB cell bodies is not observed when a BDNF sense-strand control cRNA is used (C), indicating that the antisense cRNA probe for BDNF mRNA binds specifically. trkB mRNA is also expressed by all SNB (D) and RDLN (E) motoneurons. trkB sense-strand control cRNA confirms that the antisense trkB probe binds specifically (F). Scale bars, 5 µm.

 
Androgens regulate BDNF and trkB mRNA levels in SNB, but not RDLN, motoneurons
Two-tailed t tests revealed that the amount of BDNF mRNA in SNB motoneurons decreased significantly 14 d after castration (GDX) compared with gonadally intact males [SHX; Fig. 2AGo; P < 0.006, two-tailed; t = 3.753; degrees of freedom (df) = 12]. In contrast, t tests showed that removal of gonadal androgens had no effect on BDNF mRNA levels in RDLN motoneurons (Fig. 2BGo; P > 0.16, two-tailed; t = 1.47; df = 12). In general, RDLN motoneurons contained slightly higher BDNF mRNA levels than did SNB motoneurons.


Figure 2
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  		FIG. 2. Effect of castration on BDNF and trkB mRNA levels in SNB and RDLN motoneurons. Two-tailed t tests show BDNF mRNA levels are significantly decreased in SNB (A), but not RDLN (B) motoneurons 14 d after castration (GDX) compared with Sham GDX (SHX) males. Two-tailed t tests show trkB mRNA levels are also significantly decreased in SNB (C) but not RDLN (D) motoneurons of GDX males compared with those of SHX males. Values are means (±SEM) of seven males per treatment group. **, GDX group is significantly different from Sham GDX (P < 0.006). *, GDX group is significantly different from Sham GDX (P < 0.041).

 
Levels of trkB mRNA in SNB motoneurons also decreased significantly 14 d after castration compared with gonadally intact male rats (Fig. 2CGo; P < 0.041, two-tailed; t = 2.291; df = 12). Similar to the patterns observed for BDNF mRNA, castration had no apparent effect on trkB mRNA levels in RDLN motoneurons (Fig. 2DGo; P > 0.601, two-tailed; t = 0.5382; df = 12). As with BDNF mRNA expression, levels of trkB were slightly higher in RDLN motoneurons than SNB motoneurons in SHX males.

Androgens regulate the expression of BDNF protein in SNB, but not RDLN, dendrites
We used triple-label confocal microscopy to characterize the distribution of BDNF protein in Fluorogold-labeled SNB and RDLN motoneuronal dendrites and their glutamatergic afferents (containing VGLUT1 protein). In all treatment groups, Fluorogold labeling was present in the proximal dendrites of SNB and RDLN motoneurons, and the length of dendrite labeled by Fluorogold did not change among SHX, T-treated GDX, and blank-treated GDX rats (Table 1AGo). These dendritic lengths labeled with Fluorogold represent approximately 10% of the total dendritic length of SNB dendrites revealed by the retrograde tracer cholera toxin-horseradish peroxidase conjugate (5), thus limiting our analysis to only the proximal aspect of the dendritic arbor. Despite this limitation, BDNF immunoreactivity in all groups was confined to SNB and RDLN dendrites labeled by Fluorogold and were never observed in the more distal aspects of the dendrites. Moreover, BDNF was present in all Fluorogold-labeled dendrites of SNB and RDLN motoneurons in all groups with one exception, SNB motoneurons from blank-treated castrates. Although BDNF immunoreactivity was present in the somata of all SNB and RDLN motoneurons, independent of the hormonal status of the animal, the relative length of Fluorogold-labeled dendrites containing BDNF immunoreactivity changed significantly with hormone treatment for SNB motoneurons (F2,17 = 6.364; P < 0.01), decreasing in blank-treated GDX rats compared with either SHX (P < 0.05) or T-treated GDX rats (Figs. 3AGo and 4Go; P < 0.05), shown by post hoc analysis using Bonferroni-adjusted t tests. Such retraction of BDNF from proximal SNB dendrites after castration was not observed in RDLN motoneurons (Figs. 3BGo and 5Go; F2,17 = 0.289; P < 0.753).


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  		TABLE 1. Length of Fluorogold-labeled dendrite, number of VGLUT1-positive appositions, and percentage of VGLUT1-positive appositions also positive for trkB

 

Figure 3
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  		FIG. 3. Effects of castration on BDNF protein in the proximal dendrites of SNB and RDLN motoneurons. A, One-way ANOVA shows the percentage of Fluorogold-labeled SNB dendrites containing BDNF is significantly decreased in GDX blank-treated (GDX + blank) males 14 d after castration. B, In contrast, the percentage of Fluorogold-labeled RDLN dendrites containing BDNF is no different across treatment groups. Values are means (±SEM) of six males per treatment group. **, GDX + blank group is significantly different from GDX + T and SHX groups (P < 0.01).

 

Figure 4
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  		FIG. 4. Castration causes a reduction of BDNF protein in proximal SNB dendrites. A compilation of 38–43 optical sections of a Z-series (Z-thickness, 1.0 µm) shows the cell body and proximal dendrites of SNB motoneurons positive for Fluorogold (green label, top) and BDNF (red label, middle) and merged BDNF and Fluorogold (yellow, bottom) from a SHX (right column), a T-treated GDX (T + GDX, middle column), or a blank-treated GDX (a blank + GDX, left column) male rat. The merged images reveal extensive overlap of BDNF and Fluorogold (yellow) throughout the cell body and proximal dendrites of SNB motoneurons but only for gonadally intact or T-treated castrated males (SHX and T + GDX). Note that although SNB motoneurons of blank-treated castrated males still contain BDNF, their proximal dendrites are largely devoid of BDNF, in contrast to the proximal dendrites of SNB motoneurons in males that have androgens. Inset, Magnified view of proximal dendrites showing overlap of Fluorogold and BDNF (yellow) in SHX and T + GDX males and the lack of overlap in blank + GDX males (green).

 

Figure 5
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  		FIG. 5. Castration has no effect on BDNF protein distribution in RDLN motoneurons. A compilation of 37 optical sections of a Z-zeries (Z-thickness, 1.0 µm) from a blank-treated GDX male shows two RDLN cell bodies and proximal dendrites positive for Fluorogold (green label, top) and BDNF (red label, middle). Note the extensive overlap of BDNF and Fluorogold (yellow, bottom) in RDLN cell bodies and proximal dendrites when the two images are merged. Inset, Magnified view of proximal dendrites showing overlap of BDNF in Fluorogold in RDLN proximal dendrites.

 
Little to no BDNF was detected in glutamatergic (VGLUT1-positive) nerve terminals apposed to Fluorogold-labeled SNB and RDLN motoneuron dendrites. However, many of the dendrites that were apposed by glutamatergic terminals contained BDNF (Fig. 6Go). This was the case for both SNB and RDLN dendrites from all treatment groups with the exception of SNB motoneurons from blank-treated GDX males in which BDNF had retracted from the dendrites.


Figure 6
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  		FIG. 6. Glutamatergic appositions contact BDNF-containing SNB dendrites. A compilation of 40 optical sections of a Z-series (Z-thickness, 1.0 µm) through the SNB region from a T-treated castrated male shows Fluorogold (green label), BDNF (red label), and VGLUT1 (pink, pseudocolored for contrast). Merging of the three images shows that BDNF-containing dendrites are contacted by VLUT1 puncta that lack BDNF. Inset, Magnified view of BDNF containing SNB dendrites contacted by BDNF-negative but VGLUT1-positive puncta.

 
Although the total number of synaptic inputs to SNB motoneuronal somata decreases after adult castration (33), we found that the number of VGLUT1-positive appositions to dendrites was not different among SHX, T-treated GDX, and blank-treated GDX rats (Table 1BGo). Overall, the number of VGLUT1-positive appositions to Fluorogold-labeled SNB and RDLN dendrites was substantial, with a range of 3.2 ± 0.4 to 4.1 ± 0.3 terminals occurring per 10 µm of Fluorogold-labeled dendrites in both populations of motoneurons (Table 1BGo).

trkB is found in SNB and RDLN dendrites and in glutamatergic terminals apposed to SNB and RDLN motoneurons
We used triple-label confocal microscopy to examine trkB protein expression in SNB and RDLN motoneurons and in glutamatergic inputs to these motoneurons. The trkB immunoreactivity colocalized with the somata of retrogradely labeled SNB and RDLN motoneurons and had a punctate appearance presumably on the surface of these cells. Finding trkB protein in SNB and RDLN somata is consistent with our finding trkB mRNA in these motoneurons. In addition, the rotational Zeiss LSM Image Browser software revealed punctate trkB immunoreactivity on Fluorogold-labeled dendrites of SNB and RDLN motoneurons. However, unlike BDNF protein, the distribution and density of trkB immunoreactivity in SNB and RDLN motoneuronal dendrites did not change noticeably after hormone manipulations. We also found trkB immunoreactivity in many VGLUT1-positive contacts on Fluorogold-labeled SNB and RDLN proximal dendrites. The resolving power of optical sections of 1.0 µm in our Z-series was adequate to detect trkB immunofluorescence within the VGLUT1-positive puncta, which ranged in size from 0.3–0.5 µm. The proportion of glutamatergic terminals on SNB and RDLN dendrites that also contained trkB immunoreactivity did not change across treatment groups (Table 1CGo). It is noteworthy that many of the glutamatergic contacts that contained trkB were apposed to trkB-positive sites in the dendrites of both SNB and RDLN motoneurons (Fig. 7Go).


Figure 7
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  		FIG. 7. trkB protein is contained in SNB cell bodies and their proximal dendrites and in glutamatergic contacts on proximal SNB dendrites. A compilation of 42 optical sections of a Z-series (Z-thickness, 1.0 µm) from a castrated, T-treated male shows triple-label immunocytochemistry for Fluorogold (green label), trkB (red label), and VGLUT1 (blue label). Merging of the three images shows that Fluorogold-labeled SNB cell bodies and dendrites contain trkB protein (yellow) and that proximal dendrites contacted by VGLUT1 puncta also contain trkB (pink and/or white). Note the relative absence of VGLUT1 labeling of the left side of the photomicrograph that includes a portion of white matter of the ventral funiculus. Arrows indicate VGLUT1 puncta positive for trkB immunofluorescence. Arrowhead indicates a SNB dendrite positive for trkB protein contacted by a VGLUT1-containing terminal that is also positive for trkB protein. Insets, Magnified views of SNB dendrites contacted by VGLUT1 puncta containing trkB.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results provide compelling evidence that SNB and RDLN motoneurons transcribe BDNF and trkB mRNAs. Furthermore, we show that after the loss of androgens, the levels of both BDNF and trkB mRNAs decrease significantly in SNB but not RDLN motoneurons. We also found BDNF and trkB proteins in the somata and dendrites of both motoneuronal pools. Paralleling our data from ISH, BDNF protein decreased in dendrites after castration only in SNB motoneurons and not in RDLN motoneurons. Although trkB mRNA decreased in SNB motoneurons after castration, there was no discernable change in the presence or subcellular localization of the trkB protein. We also found trkB, but not BDNF, protein localized in glutamatergic afferents to these motoneurons. Although BDNF protein appeared to be lost from the proximal dendrites of SNB motoneurons after castration, glutamatergic terminals on SNB dendrites of castrates remain intact. Taken together, these findings suggest that androgens regulate BDNF gene expression in SNB motoneurons, but not RDLN motoneurons, and that one consequence of this regulation is a change in the availability of BDNF protein in SNB dendrites. Because trkB protein was often localized on SNB neurons and on their glutamatergic afferents, BDNF signaling can potentially affect SNB function via an autocrine and/or paracrine pathway as well as more traditional retrograde pathways.

What is the role of dendritic BDNF, and how might its absence affect SNB motoneurons? Past studies have suggested that BDNF from the BC/LA muscles may be required for the maintenance of SNB dendritic arbors (6, 7). However, the results of those studies are somewhat difficult to interpret because motoneurons were axotomized. Nonetheless, BDNF applied to cut axons was effective in restoring SNB dendrites, but only if animals were also treated with T. This result suggests that T may also increase the amounts of BDNF produced by the motoneurons themselves, which when added to the BDNF obtained distally is sufficient to maintain the length SNB dendrites. Our results indicating that BDNF expression is up-regulated by androgens in SNB motoneurons are consistent with this suggestion. Because castration, like axotomy, causes SNB dendrites to regress, decreasing the level of BDNF mRNA and/or BDNF protein in SNB motoneurons after castration may lead to the same result as axotomy through comparable BDNF-dependent mechanisms.

Other model systems also provide persuasive evidence that BDNF may be actively involved in maintaining neuronal dendrites and their synapses through selective trafficking and local action in dendrites (31, 50, 51, 52). Interestingly, dendritic targeting of BDNF mRNA and protein requires prior BDNF signaling via the trkB receptor in hippocampal neurons (51). In addition, BDNF can act as an autocrine signal to influence synthesis of dendritic proteins and postsynaptic-associated proteins (53, 54). Autocrine activity of dendritic BDNF occurs via activation of its receptor at the same dendrites, which in turn, phosphorylates postsynaptic N-methyl-D-aspartate (NMDA)-subtype receptors and enhances excitatory signaling (55, 56, 57). BDNF may act via similar pathways in SNB motoneurons to maintain their dendrites and synapses, given that they have glutamatergic inputs, pre- and postsynaptic trkB receptors, NMDA receptors (28), and BDNF-containing dendrites. Moreover, many of these components are influenced by androgens in the SNB system (1, 4, 28, 33).

Because BDNF found in neurons can come from a postsynaptic target (18, 19, 20), it is difficult to delineate the origin of BDNF protein that is present in SNB motoneurons (58). However, because we find that SNB motoneurons also express mRNA for BDNF, it seems likely that some portion of BDNF protein in SNB motoneurons is produced in the motoneurons themselves. We also find that expression of BDNF mRNA in SNB motoneurons is regulated by androgens, with levels decreasing after castration. Because BDNF also influences the level of androgen receptor (AR) expression in SNB motoneurons, these results provide new evidence of a reciprocal relationship between AR function and BDNF. Past studies show that axotomy of SNB motoneurons causes a reduction of nuclear AR protein, which is reversed when BDNF is applied to the distal end of the severed axon (6). We now show that a loss of AR activity due to castration causes a reduction of both BDNF and trkB mRNA in SNB motoneurons. Furthermore, we find that BDNF protein retracts from SNB dendrites after castration. Thus, activation of nuclear AR in SNB motoneurons may influence BDNF and/or trkB gene expression, thereby changing the distribution of BDNF, and possibly trkB, protein within SNB motoneurons. It is currently unknown whether androgen acts directly on SNB motoneurons to regulate BDNF mRNA and/or protein expression or whether AR regulates the BDNF gene through some other cell type. In contrast to the recent findings of others (59), we did not detect a change in levels of trkB protein in SNB motoneurons after castration. Although it is possible that our method was not sufficiently sensitive, it may be that changes in trkB protein, as opposed to mRNA, require a longer delay after castration. Thus, changes in trkB protein that are observed 28 d after castration (59), may not be occurring or detectable in animal castrated for 14 d as in our study. In addition, because half-lives of proteins can vary greatly, it is possible that such a change in trkB protein can occur well after the change in trkB mRNA we observed.

We find a close relationship between BDNF, trkB, and excitatory glutamatergic inputs to SNB and RDLN dendrites. For example, in gonadally intact animals, we found many examples of BDNF-containing SNB and RDLN dendrites that had closely apposed glutamatergic terminals. Likewise, we also observed many glutamatergic/trkB-positive terminals that apposed BDNF-containing SNB and RDLN dendrites in gonadally intact and androgen-treated animals. Overall, we found 90–125 glutamatergic contacts apposed to Fluorogold-labeled proximal dendrites of each SNB and RDLN motoneuron. Of these terminals, about 80% were also positive for trkB immunoreactivity. In many systems, BDNF is released from presynaptic glutamatergic terminals (16, 52, 60). However, although BDNF is abundant in motoneuronal dendrites, we found little evidence of BDNF in presynaptic glutamatergic afferents. We also found that many of these trkB/VGLUT1-postive puncta that were contacting SNB and RDLN dendrites were themselves trkB immunopositive. This arrangement of trkB in both presynaptic glutamatergic terminals and postsynaptic BDNF-containing SNB dendrites suggests that BDNF may act both as a retrograde messenger to influence glutamatergic afferents to SNB and RDLN motoneurons and as an autocrine signal that affects SNB and RDLN motoneurons themselves (see Fig. 8Go for a model). Notably, the potential for retrograde and/or autocrine signaling of dendritic BDNF is apparently diminished for SNB motoneurons after castration. Although we detect a decrease in BDNF protein only in SNB dendrites after castration, it is possible that BDNF levels also decrease in the cell body. Nonetheless, any reduction in dendritic BDNF is likely to perturb BDNF-dependent signaling pathways that may be involved in dendritic maintenance. One interesting question to pursue in the future is whether a reduction in motoneuronal BDNF, either endogenously produced by motoneurons or exogenously provided by the target muscles, will trigger retraction of SNB dendrites, independent of androgens. Because RDLN motoneurons also abundantly express BDNF, we might also expect that a reduction of BDNF in RDLN motoneurons may also trigger their dendrites to retract as well. Such a finding would lend considerable credence to the idea that BDNF plays a crucial role in maintaining motoneuronal dendritic arbors.


Figure 8
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  		FIG. 8. Two potential BDNF signaling pathways for maintenance of glutamatergic synapses on SNB dendrites. BDNF in SNB dendrites (1 ) can be released and act in an autocrine fashion (2 ) to activate trkB receptors (3 ), which phosphorylate NMDA receptors (4 ) and enhance excitatory signaling. BDNF in SNB dendrites can also act as a retrograde molecule (A) to bind trkB receptors on afferent glutamatergic appositions (B), thereby activating presynaptic trkB receptors to enhance glutamate release (C) subsequently activating glutamate receptors postsynaptically (D). Prolonged NMDA receptor signaling can lead to the recruitment of postsynaptic density proteins (5 or E) to strengthen synapses and maintain dendritic integrity. Because trkB protein is localized both in SNB dendrites and in their afferents, these two potential pathways are not mutually exclusive.

 
It is possible that we have identified one of the underlying causes triggering dendritic retraction after androgen deprivation. Distinct reductions in SNB soma size, dendritic arborization, and synaptic contacts have been recorded from 25–72 d after castration (4, 7, 33, 34, 35). Dendritic BDNF and BNDF mRNA was reduced in SNB motoneurons 14 d after castration, indicating that the phenomenon may precede and possibly cause changes in SNB morphology after castration. Supporting this suggestion, we observed no loss of glutamatergic contacts apposed to SNB dendrites in our castrates. However, it is important to keep in mind that previous reports of a loss of synapses on SNB motoneurons after castration (33) may not involve VGLUT1-containing glutamatergic terminals at all but may reflect a reduction of other excitatory or inhibitory terminals not visualized in this study. Furthermore, a loss of synaptic inputs may occur within only a few days after castration because only 2 d of T treatment is sufficient to increase the number of synapses to SNB motoneurons of castrates (61). Future time-course experiments will be needed delineate the order of changes in dendritic and synaptic morphology that follows a loss of androgens.

In conclusion, these data suggest a potential mechanism by which androgens may act via BDNF and trkB signaling to maintain the highly elaborate dendritic arbor of SNB motoneurons and their afferent synaptic inputs. BDNF and trkB gene expression in SNB motoneurons depends on androgens, and dendritic localization of BDNF protein is also androgen dependent. Our data are consistent with the hypothesis that dendritic BDNF has both retrograde and autocrine effects. BDNF could act as a retrograde signal via activation of trkB receptors found on presynaptic glutamatergic terminals and/or as an autocrine signal to activate trkB receptors on SNB motoneurons themselves. Although this system appears to remain intact in RDLN motoneurons after castration, it requires androgens for its maintenance in SNB motoneurons. The loss of dendritic BDNF in SNB motoneurons may be an early event leading to the loss of dendrites and synapses that SNB, but not RDLN, motoneurons experience after castration.


   Footnotes
 
This work was supported by National Institutes of Health Grant NS28421 to S.M.B. and by NIH Grant NS045195 to C.L.J.

First Published Online April 26, 2007

Abbreviations: AR, Androgen receptor; BC, bulbocavernosus; BDNF, brain-derived neurotrophic factor; df, degrees of freedom; GDX, gonadectomized; ISH, in situ hybridization; LA, levator ani; NMDA, N-methyl-D-aspartate; RDLN, retrodorsolateral nucleus; SHX, sham gonadectomized; SNB, spinal nucleus of the bulbocavernosus; SSC, standard saline citrate solution; T, testosterone; trkB, tyrosine kinase B; VGLUT1, vesicular glutamate transporter 1.

Received March 6, 2007.

Accepted for publication April 18, 2007.


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 Discussion
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